The job of our immune system is to recognize foreign invaders and eliminate the threat while ignoring host tissues. Tissue microenvironment plays a critical role in orchestrating the generation of immune responses, including adaptive memory T cell immunity. Understanding how tissue microenvironment affects the balance between immune activation and unresponsiveness in vivo is crucial in defining pathogenesis of diseases such as cancer. It is also helpful in taking advantage of the exquisite sensitivity and specificity of the immune system in developing the next generation of therapeutic and vaccination strategies. Recently application of intravital 2-photon laser scanning microscopy in examining immune tissue organization and immune cellular orchestration in vivo has yielded new insights into the critical contribution of inflammatory chemokines in orchestrating efficient cell-cell cooperation among various arms of the adaptive immune response. Using animal tumor models genetically engineered to secrete inflammatory chemokines such as CCL3 and CCL4, we propose to undertake a series of studies to investigate how efficient cell-cell interaction among tumor-associated antigen presenting cells, CD4+T cells, CD8+ T cells, and tumor cells may be achieved to accomplish efficacious generation of long-term, protective anti-tumor T cell responses. We also outline an ambitious goal to directly observe, for the first time, dynamic cellular interplay in metastatic lymph nodes including direct antigen presentation by tumors and cross-presentation of tumor-associated antigens by host antigen-presenting cells during the induction of an effective immune response. This will be accomplished by utilizing the state-of-the-art 2-photon laser scanning microscopy available in our own laboratory. Success in this endeavor not only will yield new insights into how to employ inflammatory chemokines as an adjuvant in the generation of anti-tumor CD8+ memory T cells, but also will provide direct in vivo evidence for the roles which the host APC and tumors play in priming CD8+ T cells against tumor-associated antigen. A detailed understanding of the intricate cellular recruitment and communication in the tumor microenvironment will allow the development of targeted therapies designed to enhance anti-tumor immune activation. It will also provide insights into factors that will enhance targeting and recruitment of cytotoxic anti-tumor lymphocytes to tumor sites in currently existing immunotherapy protocols. PUBLIC HEALTH RELEVANCE: Ultimately, any effective cellular immunotherapy against cancer must accomplish 2 goals: 1) Finding effective adjuvant to enhance efficient generation and maintenance of long-term anti-tumor immunity; 2) Proper and rapid recruitment of relevant immune cells to sites of tumor-antigen priming, activation and effector delivery. This study seeks to investigate the cellular mechanism of enhanced anti-tumor effect by inflammatory chemokines such as CCL3 and CCL4, and the associated chemokine receptor(s). We propose to employ the state-of-the-art 2-photon laser scanning microscopy technology in order to accomplish this goal. Successful execution of this project will not only provide new insights into novel tumor immunotherapy approaches, but will also demonstrate a new in situ experimental platform to test future anti-tumor immune strategies with high cellular specificity.